Modeling of the chemistry of quinoprotein methanol dehydrogenase. Oxidation of methanol by calcium complex of coenzyme PQQ via addition-elimination mechanism

Full text of DOI:10.1021/ja963366d

J. Am. Chem. Soc. 1997, 119, 439-440
Modeling of the Chemistry of Quinoprotein
Scheme 1
439
Methanol Dehydrogenase. Oxidation of Methanol
by Calcium Complex of Coenzyme PQQ via
Addition-Elimination Mechanism
Shinobu Itoh,* Hirokatsu Kawakami, and
Shunichi Fukuzumi*
PQQ derivative efficiently oxidizes methanol to formaldehyde
Via an addition-elimination mechanism.
1
0
Department of Applied Chemistry, Faculty of
When the trimethyl ester of PQQ (PQQTME) was treated
with Ca(ClO4)2 in anhydrous acetonitrile (CH3CN), the absorp-
tion band at 354 nm due to the quinone shifted to 368 nm and
the shoulder around 280 nm decreases with clear isosbestic
Engineering, Osaka UniVersity
-1 Yamada-oka, Suita, Osaka 565, Japan
2
ReceiVed September 25, 1996
1
1
points at 268, 289, 303, 361, and 422 nm. The 1:1 complex
2
+
formation between PQQTME and Ca
with the binding
Bacterial methanol dehydrogenase (MDH, E.C. 1.1.99.8) is
a quinoprotein that involves a heterocyclic o-quinone cofactor
PQQ (pyrroloquinolinequinone) as a redox catalyst for the
-
1
constant Kc of 1900 M has been determined by analyzing the
11
1
13
spectral change. The following H- and C-NMR data in CD3-
2
+
oxidation of methanol to formaldehyde.1,2 The most curious
CN indicate that the binding position of Ca to PQQTME in
solution is the same as that to PQQ in the enzymatic system
1H in Scheme 1). In the H-NMR spectra, the methyl ester
1
2
1
(
protons at the 7-position move toward downfield more than
those at the 2- and 9-positions by the complexation with Ca2
+
(
∆δ ) +0.14, +0.02, and +0.06, respectively) and the ∆δ
(
downfield shift by the complexation) value of H-8 is also larger
than that of H-3 (+0.09 and +0.07, respectively). In the C-
11
13
NMR spectra, C-5 and C-7′ (ester carbonyl carbon at the
7
-position) shifted downfield (∆ppm ) +2.0 and +2.8,
feature of this enzyme for chemists may be the high reactivity
toward methanol, the most inert alcohol.3 In other words, how
does MDH activate PQQ to undergo such a difficult oxidation
respectively), while C-4, C-2′ (ester carbonyl carbon at the
2-position), and C-9′ (ester carbonyl carbon at the 9-position)
shifted upfield by the complexation with Ca (∆ppm ) -1.0,
-0.2, and -1.0, respectively).
2
+
4
11
reaction? Recently, the crystal structure of MDH from
2+
methylotrophic bacteria has been determined by two independent
research groups to provide full particulars of the enzyme active
center. According to the reported X-ray structure, there is one
calcium ion strongly bound to PQQ through its C-5 quinone
Addition of methanol into a CH CN solution of Ca -complex
3
1H resulted in a spectral change corresponding to the C-5
hemiacetal formation (2 in Scheme 1). The formation constant
K
M
of Ca (0.63 M ),
with Ca
Coordinative interaction of methanol to Ca may also enhance
the nucleophilicity of methanol by lowering the pKa value of
the -OH group.
13
2
+
add
of the Ca -complex with methanol was determined as 3.6
which is six times larger than that measured in the absence
-
1
carbonyl oxygen, N-6 pyridine nitrogen, and C-7 carboxylate
group in the enzyme active site.5
-7
Davidson and his co-
2+
-1 11,13
indicating clearly that the complexation
enhances the stability of the C-5 hemiacetal.
workers have recently suggested that Ca2 plays an important
role for the structural stabilization of the enzyme. However,
little is known about the catalytic role of Ca for the redox
reaction in MDH.9 In this communication, we present the first
functional model for MDH, where the calcium complex of a
+
2+
8
2+
2+
To our surprise, addition of methanol into a deaerated CH3-
2+
CN solution of the Ca -complex in the presence of a base such
as DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) resulted in forma-
tion of reduced PQQTME (Figure 1). The final spectrum of
the reaction mixture is essentially the same as that obtained by
the treatment of authentic PQQTMEH2 (quinol form)14 with Ca-
(
1) Salisbury, S. A.; Forrest, H. S.; Cruse, W. B. T.; Kennard, O. Nature
979, 280, 843.
2) Anthony, C; Ghosh, M.; Blake, C. C. F. Biochem. J. 1994, 304, 665,
and references cited therein.
3) Methanol can be used as a solvent for oxidation reactions by highly
1
(
(
oxidation-active quinones such as DDQ (2,3-dichloro-5,6-dicyano-1,4-
benzoquinone) and chloranil (tetrachlorobenzoquinone), see: Becker, H.-
D. In The chemistry of the quinonoid compounds; Patai, S., Ed.; John Wiley
(
ClO4)2 and DBU in deaerated CH3CN, and, more importantly,
it is also Very close to the absorption spectrum of fully reduced
MDH.
1
5
&
Sons: New York, 1974; Part 1, pp 335-424.
From the reaction mixture in a preparative scale
(
4) The two-electron redox potential of free PQQ (-0.175 V vs SCE at
3
pH 7.0) is much lower than that of DDQ and chloranil, see: Kano, K.;
Mori, K.; Uno, B.; Kubota, T.; Ikeda, T.; Senda, M. Bioelectrochem.
Bioenerg. 1990, 23, 227.
(10) An addition-elimination mechanism through a hemiacetal inter-
mediate and an acid-base catalyzed hydride transfer mechanism have been
proposed for the MDH-catalyzed oxidation of methanol,2 but no direct
evidence to support such possibilities has so far been reported.
(11) See Supporting Information.
(12) In spite of our great efforts, a single crystal of the Ca complex
suitable for the X-ray analysis could not be obtained. However, the binding
position of Ca2 to PQQTME has been confirmed by comparing the Kc
values and the spectral data of other PQQ model compounds, see: (a) Itoh,
S.; Huang, X.; Kawakami, H.; Komatsu, M.; Ohshiro, Y.; Fukuzumi, S. J.
Chem. Soc., Chem. Commun. 1995, 2077. (b) Itoh, S.; Kato, J.; Inoue, T.;
Kitamura, Y.; Komatsu, M.; Ohshiro, Y. Synthesis, 1987, 1067. (c) Itoh,
S.; Fukui, Y.; Haranou, S.; Ogino, M.; Komatsu, M.; Ohshiro, Y. J. Org.
Chem. 1992, 57, 4452. (d) Itoh, S.; Fukui, Y.; Ogino, M.; Haranou, S.;
Komatsu, M.; Ohshiro, Y. J. Org. Chem. 1992, 57, 2788.
(13) Itoh, S.; Ogino, M.; Fukui, Y.; Murao, H.; Komatsu, M.; Ohshiro,
Y.; Inoue, T.; Kai, Y.; Kasai, N. J. Am. Chem. Soc. 1993, 115, 9960.
(14) Itoh, S.; Ohshiro, Y.; Agawa, T. Bull. Chem. Soc. Jpn. 1986, 59,
1911.
(15) (a) Frank, J.; Dijkstra, M.; Duine, J. A.; Balny, C. Eur. J. Biochem.
1988, 174, 331. (b) Richardson, I. W.; Anthony, C. Biochem. J. 1992, 287,
709. (c) Harris, T. K.; Davidson, V. L. Biochemistry 1993, 32, 4362.
(
5) (a) White, S.; Boyd, G.; Mathews, F. S.; Xia, Z.-x.; Dai, W.-w.;
Zhang, Y.-f.; Davidson, V. L. Biochemistry 1993, 32, 12955. (b) Xia, Z.-
x.; Dai, W.-w.; Zhang, Y.-f.; White, S. A.; Boyd, G. D.; Mathews, F. S. J.
Mol. Biol. 1996, 259, 480.
2
+
+
(6) (a) Blake, C. C. F.; Ghosh, M.; Harlos, K.; Avezoux, A.; Anthony,
C. Nature Struct. Biol. 1994, 1, 102. (b) Ghosh, M.; Anthony, C.; Harlos,
K.; Goodwin, M. G.; Blake, C. Structure 1995, 3, 177.
2
+
(
7) The presence of Ca in the enzyme active site has been also
suggested for other PQQ-dependent enzymes such as ethanol dehydrogenase
from Pseudomonas aeruginosa and glucose dehydrogenase from Acineto-
bacter calcoaceticus, see: (a) Mutzel, A.; G o¨ risch, H. Agric. Biol. Chem.
1
991, 55, 1721, (b) Geiger, O.; G o¨ risch, H. Biochem. J. 1989, 261, 415.
(
8) Harris, T. K.; Davidson, V. L. Biochem. J. 1994, 303, 141.
2
+
(
9) Ca in enzymatic systems has been generally believed to act as
information mediators, enzyme-structure stabilizers, and enzyme-activity
regulators, but little is known about its catalytic role in enzymatic redox
reactions, see: Kaim, W.; Schwederski, B. Bioinorganic Chemistry:
Inorganic Elements in the Chemistry of Life; John Wiley & Sons: New
York, 1994.
S0002-7863(96)03366-5 CCC: $14.00 © 1997 American Chemical Society

440 J. Am. Chem. Soc., Vol. 119, No. 2, 1997
Communications to the Editor
-
3
Figure 2. Plot of (A) kobs vs [CH
3
OH] (at [DBU] ) 2.4 × 10 M)
and (B) kobs vs [DBU] (at [CH OH] ) 1.5 M) for the oxidation of
3
-
5
CH
(
3
OH by PQQTME (2.5 × 10 M) in the presence of Ca(ClO
4 2
)
Figure 1. Spectral change observed in the oxidation of CH
3
OH (2.2
-3
6.3 × 10 M) and DBU in deaerated CH
3
CN at 298 K. The solid
-
5
M) by PQQTME (2.5 × 10 M) in the presence of Ca(ClO
4
)
2
(6.3 ×
lines are drawn based on the computer simulation using the kinetic
equation and the parameters indicated in the text.
-
3
-3
1
0
M) and DBU (2.4 × 10 M) in deaerated CH
3
CN at 298 K.
-
4
-3
(
[
[PQQTME] ) 1.5 × 10 M, [Ca(ClO4)2] ) 8.5 × 10 M,
Scheme 2
-
3
DBU] ) 2.4 × 10 M, [CH3OH] ) 2.2 M in 100 mL of
CH3CN), a reasonable amount of formaldehyde was isolated
as the 2,4-dinitrophenylhydrazone derivative.16 A similar
spectral change (quantitative formation of reduced PQQTME)
was observed with ethanol as well as methanol, and the
oxidation product, acetaldehyde, was obtained quantitatively as
2
,4-dinitrophenylhydrazone derivative. Furthermore, the oxida-
intermolecular hydride transfer mechanism from methanol to
the quinone. A large kinetic isotope effect of 6.4 on the rate
constant k was obtained by using CD3OD as a substrate, clearly
indicating that the base-catalyzed R-proton abstraction from the
substrate is rate-determining in the methanol oxidation reaction
tion of ethanol to acetaldehyde proceeded catalytically (1450%
based on PQQTME after 65 h), when the reaction was carried
out under aerobic conditions. It should be noted that the
presence of both Ca2 and DBU is essential for the redox
reaction between PQQTME and the alcohols to occur in our
model system.
+
(from 2 to 3).
In summary, we have demonstrated here that the Ca2+-
The pseudo-first-order rate constant (kobs) obtained from the
spectral change indicated in Figure 1 shows a Michaelis-
Menten type saturation phenomenon when plotted against
methanol concentration (Figure 2A).17 Nonlinear curve fitting
using the equation of kobs ) kKadd[CH3OH][DBU]/(1 + Ka[D-
BU] + Kadd[CH3OH]) derived from the reaction mechanism
shown in Scheme 2 provided the kinetic parameters as Ka )
complex of PQQTME actually facilitates the alcohol-adduct
formation at C-5 and also that Ca is required for the base-
catalyzed oxidative elimination reaction of the adduct. In the
2+
enzymatic system, aspartate (Asp) is suggested to be the most
5,6
likely candidate as the general-base catalyst.
Alternatively,
intramolecular general-base catalysis by the C-4 carbonyl
oxygen could be expected to abstract the R-proton of the
substrate. Such a possibility will be examined by using other
PQQ-model compounds.
3
-1
-1
-1 -1
1
.4 × 10 M , Kadd ) 3.7 M , and k ) 0.42 M s , where
-
Ka is the deprotonation equilibrium constant (1 represents
deprotonated PQQTME at N-1) and k is the rate constant of
the redox reaction from 2 to 3. Dependence of kobs vs [DBU]
Acknowledgment. The present study was financially supported in
part by a Grant-in-Aid for Scientific Research on Priority Area
(08249223) and a Grant-in-Aid for General Scientific Research
(08458177) from the Ministry of Education, Science, Sports, and
Culture of Japan. We also thank Takeda Science Foundation for the
financial support.
(
Figure 2B) also afforded a saturation phenomenon as expected
3
-1
from the kinetic equation, from which Ka ) 1.5 × 10 M ,
-
1
-1 -1
Kadd ) 3.5 M , and k ) 0.42 M
s
were obtained by the
computer simulation. The good agreements of those kinetic
parameters determined independently from kobs vs [CH3OH] and
kobs vs [DBU] together with the agreement of Kadd determined
Supporting Information Available: Spectral change of the titration
-1
-1
by the titration (3.6 M ) and kinetics (3.5 and 3.7 M ) support
the validity of the proposed mechanism and exclude the
of PQQTME with Ca(ClO
4 2 3
) in anhydrous CH CN (S1) and its
1
13
analytical equations and results (S2), H- and C-NMR data of
2+
PQQTME and its Ca -complex in CD
3
CN (S3), and spectral change
(16) The isolated yield (20%) was just the same as that obtained by the
of the titration of Ca2 -PQQTME complex with methanol in CH
+
3
CN
reaction of authentic sample of formaldehyde with 2,4-dinitrophenylhydra-
zine under the same experimental conditions. This result indicate that
formaldehyde is formed quantitatively as in the case of acetaldehyde.
(S4) and its analytical results (S5) (5 pages). See any current masthead
page for ordering and Internet access instructions.
(
17) The rate of formation of 3 was monitored after the equilibrium in
Scheme 2 was established.
JA963366D